Organisms living in the nature are exposed to various environmental stresses such as salt stress, high temperature stress, low temperature stress, freezing stress, or drought stress. Specifically, salt stress is one of the main factors inhibiting the growth of many species of higher plants. Since the improvement of tolerance against salt in higher plants leads to the increase of the farm products, attempts have recently been made vigorously to improve salt tolerance of higher plants by gene introduction.
For example, H. J. Bohnert et al. show that salt tolerance of tobacco plants was improved by introducing the mannitol synthetase derived from coliforms into tobacco plants (Science 259, 22, 508–510, 1993). It has been shown that similar effects of improvement of salt tolerance in higher plants can be obtained by introducing proline synthetase (Plant Physiol. 108, 1387–1394, 1995) or glycine betaine synthetase (Plant J. 12, 133–142, 1997, Plant Mol. Biol. 38, 1011–1019, 1998). However, the recombinant plants obtained by the introduction of the genes encoding the enzymes will not acquire salt tolerance enough to cope with the level of the seawater.
In general, environmental stresses such as salt stress affect on various organic responses. In other words, in order to produce genetically modified plants capable of growing in an environment at a high level of salt concentration in a stable manner, it is necessary to use gene population encoding proteins having the activity of improving salt tolerance. Main methods used in the past to isolate gene population encoding proteins having the activity of improving environmental stress tolerance have been based on the assumption that “mechanisms resisting to stress express when stress is imposed.” More specifically, it detects proteins or mRNA which is specifically expressed when some environmental stress is imposed on a plant, acquires the genes for them following methods of molecular biology, and genetically introducing them to plants weak to such stress, and examines whether the plants become to show tolerance to environmental stress. It is certain that such methods have been used to isolate the genes specifically induced by environmental stress. However, it was rare that plants with a high level of tolerance to environmental stress were made by introducing the genes to plants weak to environment stress. For example, if a gene responsible for salt tolerance expresses with or without stress in a plant growing under a stressful condition such as a high concentration of salt, it is impossible to detect stress tolerance genes in the previous methods. Genome projects have recently been carried out on plants with a high level of stress tolerance. Although their base sequences or sequences of amino acids may be revealed, the present state is that as in other genome projects, there are many proteins whose functions are not identified, and it cannot specify which proteins are responsible for tolerance to environmental stress.
On the other hand, mangroves are woody plants growing in soil containing a high concentration of salt along the coast and near the entry of rivers. Mangrove plants are thought to have acquired special mechanisms for salt tolerance in the process of evolution. Therefore, if we can isolate gene population of mangrove plants responsible for salt tolerance, it is expected that the isolates can be used to apply to improve salt tolerance of higher plants. However, there is no known example of analyzing mechanism of salt tolerance of mangrove plants at genetic level. One of the reasons is that it was quite difficult to extract mRNA of the genes directly involved in salt tolerance from such woody plants.
Recently, Mimura et al. have established cultured cell lines of Bruguiera sexangula, a kind of mangrove plants (J. Res. 110, 25–29, 1997). The cultured cells are different from other cultured plant cells for their quite specific properties; they can be subjected to suspension culture, and they can grow in a stable manner under the circumstance where the salt concentration is 150 mM or more (J. Plant Res. 110, 31–36, 1997). However, it has never been attempted even to detect a group of genes involved in salt tolerance of mangrove plants by constructing cDNA library of mangrove plants with the use of such cultured cells. Moreover, there are few examples of improving salt tolerance of higher plants by introducing the genes derived from other plants with salt tolerance. One representative example is to improve salt tolerance of tobacco plants by introducing inositole methyltransferase genes derived from Masembryanthenum crystallinum, which is a halophyte, into tobacco, and increasing the content of ononitol, a kind of compatible solute, into transformed cells (Plant Physiol. 115, 1221–1219, 1998), and another example is to slightly improve salt tolerance of rice plants by introducing the genes encoding stress inducing proteins (LEA proteins) derived from barley with a relatively high level of salt tolerance, into rice plants (Plant Physiol. 110, 249–257, 1996). As shown above, there is no established technology for isolating effectively a group of genes encoding proteins having the activity of improving tolerance to salt stress, and at the present situation, the environmental stress tolerant genes in many halophytes such as a group of mangrove plants have not been studied well enough.
Further, the functions of improving salt stress tolerance of proteins can be improved by artificially modifying genes encoding the proteins having the activity of improving salt stress tolerance, it becomes possible to produce plants with a higher level of tolerance to salt stress. There was an attempt to stabilize the expression level of choline dehydrogenase in plants by modifying some codons when expressing choline dehydrogenase derived from coliforms in a plant, which leads to stabilization of the level of glycine betaine (a kind of compatible solute, which has a function of improving salt tolerance in plants), which is a metabolite of choline dehydrogenase (Stress responses of photosynthesis organisms (ed. Satoh K., Murata N.), 115–131, Elsevier Science, Amsterdam). However, this is not the one that changes a sequence of amino acids in proteins. It has never been reported to improve the level of salt stress tolerance of higher plants by introducing proteins whose sequences of amino acids are modified (improved) and whose activity improves salt stress tolerance. Further, it is expected that there are possibilities that the genes or their modified genes involved in salt tolerance have the activity of improving tolerance not only to salt stress but also to all or some of the other kinds of environmental stresses (thermal, freezing, osmotic pressure, drought, and ultraviolet).